Liquid chromatography-mass spectrometry was one of the most powerful methods for identification and detection of chemical structures of lipids. In this study, the cerebrosides molecular species from the sea cucumber Pearsonothria graeffei (P. graeffei) were high throughput identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS). Cerebrosides were separated and purified by solid-phase extraction with chloroform-methanol solution. Precursor ion scan spectra and product ion scan spectra were obtained through auto MS/MS analysis in the positive scan. Cerebroside molecules were selected according to the neutral loss fragments of 180 Da, and then the structures were identified according to pairs of specific products of sphingoid bases and their precursor ions. Eighty-nine cerebrosides molecular species were identified, large amounts of d17:1-C22:0h, t17:0-C24:1h, d17:1-C24:1h, d17:1-C23:0h, d17:1-C22:0 and d17:1-C23:0 were present which have hardly found in mammal. There were 13 classes of long-chain base (LCB), and the ratio of phytosphingosines and sphingosines was roughly 1:9, in which two of the most common LCBs were d17:1 and d18:1. The carbon numbers of fatty acids (FAs) were mainly 18~24, while 24 carbon fatty acids were predominant. The ratio of saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) was about 2:3, and the percentage of hydroxy fatty acid (HFA) was over 60%. The ratio of non-hydroxylated fatty acid (NFA)/HFA was also approximately 2:3. LC-Q-TOF-MS analysis should be useful for the structure determination of diverse cerebrosides molecular species. Meanwhile, this method provided a basis for structure-activity relationship studies and functional food development of the sea cucumber P. graeffei as well.